Therapeutic properties of oils

ABSTRACT

The present invention provides a novel scientific approach to determine whether a compound has anti-inflammatory activity. In particular, the novel assays allow the screening of compounds for the purposes of prophylactic and therapeutic use in treating or ameliorating the symptoms of T-cell, macrophage or neutrophil mediated diseases in mammals. In particular, the invention is based on the measurement of the capacity of a substance being an oil or fat, an alcoholic extract of an oil or fat, a biologically active component of an oil or a fat, or a preparation comprising an oil or fat, to suppress the activity of T-cells, macrophages or neutrophils in humans or animals in response to chemical and/or biological agents that activate these cell types. Measurements are made either in vivo (eg in mice) or in an in vitro preparation of human T-cells, macrophages or neutrophils, or a cell line derived therefrom. The substance is, in particular, emu oil or an ethanolic extract thereof. Therapeutic compositions and methods are also disclosed.

BACKGROUND OF THE INVENTION

The immune system plays a critical role in the prevention of disease andthe maintenance of health.

Diminished immune function, as occurs in the aged, in children under theage of two years, and in burns patients, as well as patients undergoingchemotherapy or transplantation, can increase the risk of disease.

On the other hand, inappropriate or excessive response of the immunesystem to infective agents or various stressors can result in tissuedamage. Accordingly, autoimmune and allergic inflammatory diseasescontinue to be a major burden to the community. These diseases resultfrom the “inappropriate” stimulation of leukocytes of the immune system,which include lymphocytes, macrophages and neutrophils. For example,chronic immune system activation can increase the risk of disease, egarthritis, cystic fibrosis, inflammatory bowel disease, Crohn's disease,graft versus host disease, multiple sclerosis (MS), systemic sclerosis,allergic contact dermatitis, psoriasis and diabetes. The main approachesto treating these diseases are to depress the immunological reactions byinhibiting a variety of responses of leukocytes (1).

There are numerous reports showing that animal and plant fats and oilshave therapeutic properties through their ability to modulate immunefunction; eg fish oils, flaxseed oil, linseed oil, borage oil, emu oiland evening primrose oil.

The Australian aboriginal practice of external application of emu oilfor treating aches and pains has provided anecdotal evidence for theanti-inflammatory properties of this oil (2,3). However, conclusivescientific evidence for the in vivo efficacy of anti-inflammatoryproperties of emu oil is lacking, with only limited studies inexperimental arthritis in rodents having been conducted thus far (4,5).It is well appreciated in the emu oil industry that theanti-inflammatory efficacy of different preparations of emu oil variessignificantly. This variation can be so significant that it hampers thetherapeutic use of this oil (6) and hence its commercial value. At themoment, no standard protocols are followed in the farming or source ofemu, the part of the bird from which the oil is obtained, the method ofpreparation or storage of emu oil (7). In fact, there are conflictingdata on the therapeutic efficacy of different emu and other oils andthere appear to be at least two reasons for this.

Firstly, most animal fats and oils are complex mixtures with highlyvariable chemical compositions. The individual components almostcertainly have different effects on immune function and may, inaddition, inhibit the activities of other components or even synergisewith each other.

Secondly, the immune system is made up of a number of different celltypes, each with highly specific roles and not all of which respond inthe same way to fats and oils. Optimum activity of an oil is thereforedependent on the condition being treated, as the cell types each havedefined roles.

Furthermore, current scientific assays and tests on the efficacy of oilshave presented conflicting results. The inability to quality control andstandardise the oil for anti-inflammatory properties has posed a majorlimitation to the use of emu oil as a therapeutic agent. Variations inthese factors can, in part, contribute to variations in the efficacy ofthe oil and have prevented its use in humans as a pharmaceutical agent,more particularly as a treatment for inflammatory diseases, conditionsor responses.

A accurate assessment of the immunosuppressive activity prior totherapeutic use would greatly increase the consistency andreproducibility of treatment with a particular oil, as well as providinga means of increasing its therapeutic activity.

Unfortunately, the prior art is lacking in methods of assessing thelikely therapeutic activity of an oil sample.

The present inventors have developed a method of measuring the intrinsiccapacity of an oil to suppress the immune system of humans and animals.The method also allows the testing of the level of therapeutic activityof an oil, thereby enabling differentiation between oil samples of lowand high therapeutic activity, and enabling oils to be graded for theirtherapeutic activity.

SUMMARY OF THE INVENTION

According to one aspect, the present invention overcomes or reduces atleast some of the above-mentioned problems by providing a novelscientific approach to accurately determine whether a compound hasanti-inflammatory activity. In particular, the novel assays allow thescreening of compounds for the purposes of prophylactic and therapeuticuse in treating or ameliorating the symptoms of T-cell, macrophage orneutrophil mediated diseases in mammals.

In particular, the invention is based on the measurement of the capacityof an oil or fat, alcoholic extracts of an oil or fat, biologicallyactive components of an oil or a fat, or preparations comprising oils orfats, to suppress the activity of T-cells, macrophages or neutrophils inhumans or animals in response to chemical and/or biological agents thatactivate these cell types. Measurements are made either in mice (ie invivo) or in human T-cells, macrophages or neutrophils isolated fromblood. The method can be used to quantify the total T-cell, macrophageand/or neutrophil suppressive activities per unit mass or volume in anyoil or fat and the degree of suppression of T-cell, macrophage orneutrophil responses by an oil or fat.

Using a model representative of a chronic inflammatory reaction (thedelayed type hypersensitivity (DTH) reaction), emu oil was found toinhibit T lymphocytes and macrophage recruitment to the site ofinflammation.

Emu oil was also found to significantly suppress the acute inflammatoryresponse induced by Carrageenan reaction. Alcoholic, and in particularethanolic, soluble fractions of emu oil were found to inhibit theability of neutrophils to adhere to endothelial cells, but in particularwere found to substantially suppress the chemotactic response ofneutrophils.

The effects of emu oil and its ethanol soluble components on T-cell,macrophage and neutrophil chemotaxis and recruitment indicate that bothemu oil and its ethanol soluble components are useful for treating acuteand chronic inflammatory reactions.

After dissolving emu oil in ethanol, the soluble fraction of emu oil(containing primarily triglycerides) was found to have anti-inflammatoryproperties and contradicts the earlier belief that emu oil by itselfdoes not have anti-inflammatory properties. The inventors haveconclusively shown that the ethanol soluble fraction of the emu oilsuppresses T-lymphocyte activity in that it suppresses bothlymphoproliferation and also the production of pro-inflammatory andpro-DTH cytokines such as interleukin-2, lymphotoxin and interferon-γ.These activities of T-lymphocytes play fundamental roles ininflammation. Further fractionation of the ethanol soluble fractionshowed that certain components contributed to anti-inflammatoryactivity, whilst others suppressed anti-inflammatory activity.

The inventors also found that the efficacy of the anti-inflammatoryproperties of the emu oil was dependent on the temperature at which theoil was rendered from emu fat. Activity was found with oils rendered attemperatures of 60° C. and 80° C., and ever better activity with oilsrendered at 100° C. However, preparations prepared at 40° C. had minimalactivity.

According to a first aspect of the invention, there is provided an assaysystem for testing samples of substances (such as emu oils and otheroils) to assess, in a standardized manner, the anti-inflammatoryactivity of each sample, and to enable different samples to be graded interms of anti-inflammatory activity (if any).

The assay system may involve administration of serially reducing amountsof the test substance (eg serially diluted in ethanol) to test animals(eg mice). Administration may be by injection (eg into the footpad), orbe intraperitoneal, topical or oral administration.

In one embodiment of the invention, the assay system comprises assessingthe anti-inflammatory activity of a compound or composition, hereinreferred to as the test substance, by

-   (i) injection of a suitable antigen into an appropriate body part    (eg footpad) of a mammal, for example a mouse;-   (ii) either injection of a predetermined amount of said test    substance into the same body part, or topical application to said    mammal of a predetermined amount of said substance;-   (iii) measurement of the degree to which swelling which would    otherwise result from injection of said antigen is reduced or    alleviated, for example in either the footpad or the immune system    organs (eg lymph nodes); and-   (iv) comparing the activity of said test substance, as measured in    step (iii), against the activity of a standard compound having known    anti-inflammatory characteristics, the activity of said standard    compound having been measured by this same assay system of steps (i)    to (iii), and having been used to generate a grading system to    compare the efficacy of various test substances.

The antigen may, for example, be Carrageenan or sheep red blood cells(SRBC), and the test substance may be an emu oil or other oil believedto have anti-inflammatory activity.

In step (i), it is preferred that the antigen is injected eitherintraperitoneally or into the footpad or ear of a mouse. In step (ii),it is preferred that the test substance is injected intraperitoneally orapplied topically.

The measurement of step (iii) is preferably undertaken some time, and inparticular about 24 hours, after injection of the test substance (step(ii)).

An alternative, in vitro assay system for testing a substance so as toassess, in a standardised manner, its anti-inflammatory activitycomprises:

-   (i) measurement of the activity of an in vitro preparation of    T-cells, macrophages or neutrophils, or a cell line derived    therefrom;-   (ii) addition of said substance to said preparation of T-cells,    macrophages or neutrophils, or said cell line derived therefrom;-   (iii) measurement of the change in activity of said preparation of    T-cells, macrophages or neurophils, or said cell line derived    therefrom, following addition of said substance in step (ii); and-   (iv) comparing the change in activity (as measured in step (iii))    for said substance against the change in activity for a standard    compound having known anti-inflammatory characteristics, the change    in activity for the standard compound having been measured by this    same assay system of steps (i) to (iii), and having been used to    generate a grading system to compare the efficacy of various test    substances.

This in vitro assay system may involve treating the preparation ofT-lymphocytes, macrophages or neutrophils, or said cell line derivedtherefrom, with serially reducing amounts of the test substance, egserially diluted in ethanol.

This assay system is a means for assessing the effect of the oil beingtested on the cell (eg T-cell, macrophage or neutrophil) mediated immuneresponse elicited by an antigen, and hence assessing itsanti-inflammatory activity.

The following are examples of the types of in vitro assays which can becarried out, according to this assay system:

-   (a) using a preparation of T lymphocytes, and measuring    lymphoproliferation;-   (b) using a preparation of T lymphocytes, and measuring their    production of cytokines, such as interleukin-2 (IL-2), tumor    necrosis factors (eg TNF α and lymphotoxin (TNF β)) and interferon γ    (IFN-γ);-   (c) using a preparation of neutrophils, and measuring their    chemotatic activity; and-   (d) using a preparation of neutrophils, and measuring their    adherence to endothelial cells.

T-cells play a major role in the tissue damage in various diseases,largely through their production of cytokines. Cytokines (such as TNF αand IL-2) produced by T-cells are believed to contribute to the tissuedamage resulting from abnormal immune function.

The use of therapeutic agents, preferably agents that are not toxic, toinhibit the production of cytokines by T-cells would be particularlyuseful in the treatment of tissue damage, particularly those mediated byT-cells.

Prior art agents used to treat T-cell mediated diseases are either toxicor have considerable systemic effects.

The present inventors have developed a method of treating or preventingtissue damage using (in particular) emu oil, a non-toxic materialproduced from the adipose tissue of emus. The inventors have developed amethod of increasing the activity of the emu oil used for this purpose,thereby ensuring reliability and consistency of the product and,moreover, have found that permeants (substances used to increase themovement of chemical substances through the skin) are not required foractivity. The inventors have also found that an alcoholic extract of emuoil so produced is also effective in treating T-cell mediated diseases.

The invention also relies on the discovery that emu oil, and alcoholicextracts of emu and other oils, are able to suppress the activity ofT-cells, being cell types that contribute to the tissue damage in avariety of human diseases. The invention involves the use of emu andother oils, as well as extracts thereof, to treat these differentdisease states by preventing or reducing the damage caused by T-cells.The use of emu oil has a further advantage in that it can also reducethe tissue damage caused by another important immune cell type, theneutrophil.

Therefore, according to a second aspect of the invention, there isprovided a composition comprising emu oil, or a biologically activeextract or component thereof, optionally together with a carriervehicle, for treating or ameliorating the symptoms of T-cell mediateddiseases or conditions or neutrophil mediated diseases or conditions inmammals. Examples of the diseases or conditions include immune complexdisease, renal disease, nephritis, arthritis (eg rheumatoid arthritis orseptic arthritis), glomerulitis, vasculitis, gout, urticaria,angioedema, cardiovascular disease, systemic lupus erythematosus, breastpain/premenstrual syndrome, asthma, neurological disease, attentiondeficit disorder (ADD), psoriasis, retinal disease, acne, sepsis,granulomatosis, inflammation, reperfusion injury, cystic fibrosis, adultrespiratory distress syndrome, thermogenesis, diabetes, inflammatorybowel disease, Crohn's disease, multiple sclerosis (MS), systemicsclerosis, osteoarthritis, atopic dermatitis, allergic contactdermatitis, graft rejection (graft versus host disease) ortransplantation.

The composition can be in the form of an oral, injectable or topicalcomposition. The biologically active extracts or components include atleast one of the following: triglyceride fractions or triglyceridefraction components, sterol fractions or sterol fraction components,phenolic fractions or phenolic fraction components, alkali-stablefractions or alkali-stable fraction components, organic solvent extracts(eg of emu oil) or components thereof. In the preferred form, theorganic solvent is ethanol.

According to a third aspect of the invention, there is provided a methodof treating or ameliorating the symptoms of T-cell mediated diseases orconditions or neutrophil mediated diseases or conditions in mammals, themethod comprising administering an effective dose of a compositioncomprising emu oil, or a biologically active extract or componentthereof (eg as exemplified above).

The composition can be administered orally, parenterally (eg byinjection) or topically.

It is preferred that said effective dose of said composition beadministered after or just before a T-cell mediated disease orcondition, neutrophil mediated disease or condition or inflammationreaction has occurred.

In a fourth aspect of the invention, an alcohol (such as ethanol), isused to extract compounds having anti-inflammatory activity from the emuoil or other biologically active oil or fat. Alternative organicsolvents which would perform the same function of solubilising andextracting effective compounds from the oil would be apparent to personsskilled in the art.

Although emu oil is specifically exemplified, it is to be understood bythose skilled in the art that the assays, methods and compositions ofthe present invention can be applied to any substance or oil of whichemu oil is but one example. Other suitable oils are, for example, otheranimal oils; plant oils, such as tea tree oil, flaxseed oil, linseedoil, borage oil or evening primrose oil; fish oils; and algal, microbialand fungal oils.

According to a fifth aspect of the invention, there is provided a methodof preparing or rendering emu oil for therapeutic use in a mammal,including the step of heating the emu oil, or the tissue from which theemu oil is derived, to a temperature of at least 40° C.

As used throughout the present specification and claims, the term“biologically active” refers to the capacity to elicit ananti-inflammatory response.

DETAILED DESCRIPTION OF THE INVENTION

The active ingredient(s) in emu oil that is (are) responsible for thereported anti-inflammatory activity has (have) not been identified. Emuoil is composed mainly of triglycerides that contain varying amounts offatty acids (Table 1). The limited available data on the composition ofemu oil suggest that the clear oil can vary markedly in terms ofanti-oxidants (carotenoids, flavonoids), skin permeation-enhancingfactors and α-linolenic acid (18:3ω3) (from 0-20%) (4) content. Thefinding that the oil is not rich in ω3 fatty acids makes it unlikelythat the anti-inflammatory effect of the oil is related to ω3 fattyacids, which are widely perceived as having anti-inflammatory actions. Aprevious study has reported, as unpublished results, that the efficacyof emu oil as an anti-inflammatory agent did not correlate with ω3 fattyacid content (0.2-19.7%) of the oil (4). TABLE 1 Fatty acid compositionof emu oil COMPONENT AMOUNT Oleic acid (18:1ω9) 47-58% Palmitic acid(16:0) 19-24% Stearic acid (18:0)  8-11% Linoleic acid (18:2ω6) 5.5-17% Hexadecenoic acid (16:1ω7)  3-6%

A combination of Thin Layer Chromatography (TLC), Gas Chromatography(GC) and Gas Chromatography-Mass Spectroscopy (GC-MS) analysesdemonstrated the presence of a wide range of fatty acids, sterols andphenols in emu oil preparations. From the TLC, it was evident thattriacylglycerol is the major component and needs to be considered as oneof the anti-inflammatory components of the oil since previous studieshave shown that fatty acids can inhibit inflammation.

In terms of its phenolic content, Makin emu oil was found to have 25μmol/l which is about 20 fold less than the level of phenols in oliveoil. Thus, it is unlikely that this is the active anti-inflammatoryelement of emu oil since olive oil has been reported not to haveanti-inflammatory properties (7) (also, our unpublished observations).

Sterol analyses revealed that emu oil was similar to tuna oil butsubstantially different from olive oil, with cholesterol making up themajor component of the emu oil sterols. The role of these substances inthe anti-inflammatory properties of emu oil was not evaluated.

The fatty acid composition of the oil was analysed independently bythree different groups using GC-MS, MS and GC. From these studies, itwas found that the major fatty acids are oleic (around 50%), palmitic(around 20%), stearic (around 10%), linoleic (around 10%) andpalmitoleic (around 5%). These could be taken as the main fatty acidcomponents of Australian emu oils. The composition of oils prepared fromemus in different geographical locations and probably prepared indifferent ways were not distinguishable based on the fatty acid contentanalyses.

Extensive studies using a standard emu oil (Makin) demonstrated that,when administered to mice, the oil consistently caused depression ofchronic and acute inflammation. For chronic inflammation, a standarddelayed type hypersensitivity reaction (DTH), which is induced andelicited by SRBC antigens, was used. The reaction was measured bymonitoring the amount of hind footpad swelling as a result of an antigenchallenge. Makin emu oil significantly inhibited the elicitation of thisinflammatory response. Since the cells involved are predominantly Tlymphocytes and macrophages, an effect, either directly or indirectly,on the accumulation of these cell types must have been caused by theadministration of emu oil. The effects of emu oil were not restricted tochronic inflammation, since it was just as effective in depressingcarrageenan-induced inflammation, considered to be a model for testingacute inflammation and which primarily involves neutrophil accumulationat the injected site.

Using the chronic inflammatory model of DTH, the effects of differentpreparations of emu oil on this response were examined in an effort toexplain the reasons for variability in the efficacy of the differentpreparations. Of the samples of emu oil examined, Makin emu oil was themost effective. Toowoomba and Little Meadow showed someanti-inflammatory activity, less was seen with emu oil A2-100G and nonewith emu oil G53. This could not be explained on the fatty acidcomposition of the emu oil samples, since these were essentially similar(Tables 5 and 6 on pages 29 to 30).

Examination of the characteristics of the depressive effects of emu oilon inflammation showed that the oil was most effective when given closeto or just after the antigen challenge. This was shown by the fact thatthe efficacy of the emu oil was greatest when the oil was given 1 hbefore, rather than 5 h before, challenge. A similar effect was shownusing the carrageenan-induced inflammation model. It was also foundthat, when the emu oil treatment was delayed to 3 h after theelicitation of the inflammatory response, the efficacy of the emu oilwas significantly more effective than treatments given 1 h beforechallenge. Firstly, this suggests that the oil acts quite rapidly oncomponents of the immune system; secondly, this shows that inflammationcan be controlled using suitably prepared emu oil even after anindividual begins to experience inflammation.

Rendering temperature was found to govern the efficacy and/or type ofoils produced since emu oil extracted at 40° C. was found to be lessactive than when it was extracted at 60° C., 80° C. or 100° C. Noevidence was found in terms of fatty acid composition by GC analysisbetween the oils produced at the latter three different temperaturessince these were very similar in content, including the levels oflinoleic acid (18:2ω6) (see, for example, Table 11 on page 40).

To identify the components in emu oil responsible for theanti-inflammatory effects, the emu oil was added directly to culturedlymphocytes and neutrophils in order to see if the activities of theseleukocytes would be altered. The studies were unsuccessful because ofthe solubility problem of the oil. To overcome this problem, the oil wassolubilised in ethanol and, following fractionation, the componentshaving anti-inflammatory activity were identified (see FIG. 28). Thesolubilised fraction had significant anti T lymphocyte activity. Since Tlymphocytes are the major cell which mediate the DTH reaction andchronic inflammation, these results show that emu oil is able tosuppress DTH activity. Chemical analysis of the ethanol fraction by GCdid not reveal any enrichment of a particular fatty acid, although therewas, however, a slight increase in the proportion of 18:2ω6. Thus, theethanol soluble fraction may be a source from which the activecomponents can be used to treat inflammation. Interestingly, the anti Tcell activity in terms of inhibition of lymphoproliferation in the emuoil preparations rendered at 40° C., 60° C. and 80° C. correlated withtheir in vivo activity with inhibition of DTH activity. The inventorshave shown that, in both instances, rendering temperature of 60°C.<T≦100° C. produces more efficacious oils than rendering at 40° C.(FIG. 15 cf FIG. 17).

Further evidence for an effect on the T cell responses was shown byexamining the effects of the ethanol soluble emu oil fraction on thecytokine products produced by activated T lymphocytes, IL-2,lymphotoxin, TNF β and IFN-γ. Production of these cytokines wasinhibited by pre-treating T lymphocytes with the solubilised emu oilfraction. The effects were extended to production of TNF by monocytesvia LPS stimulation. However, it was evident that the T cell productionof cytokines was more sensitive to emu oil than TNF production bymonocytes, showing a preferential effect of the ethanol soluble emu oilfraction for T lymphocyte responses, suggesting the T cell as a majortarget for emu oil therapy.

The solubilised fraction of Makin emu oil was found to inhibit bothchemotactic migration as well as adhesion of neutrophils to endothelialcells. Both of these properties are key functions necessary forinfiltration of neutrophils to sites of inflammation. Neutrophiladherence was also affected when endothelial cells were pre-treated withthe solubilised fraction. The combination of the effects of thesolubilised fraction on the neutrophils and endothelial cells wouldinhibit adherence of leukocytes to endothelial cells in vivo. While theeffect on neutrophils is not relevant to DTH, it is highly relevant tocarrageenan induced or acute inflammation, where the neutrophil isthought to be a key player (9).

The emu oil ethanol soluble fraction was found to be rich in free fattyacids (see Table 13 on page 45). Thus, one of the effects on Tlymphocytes could involve fatty acids such as 18:2ω6. The inventors'investigations established that serum fatty acid binding proteins suchas albumin can decrease the activity of free fatty acids by binding tothem. Further investigations were conducted as to whether or not serumcould abrogate the effects of a Makin emu oil ethanol extract, which hadbeen rendered at 40° C. The addition of serum was found to block most ofthe anti-T cell activity of this oil fraction and this would explain thediscrepancies and variations in efficacy of emu oils to treatinflammation.

On TLC separation of the ethanol soluble fraction (see FIG. 27), severaldistinct bands were seen and at least one corresponded to the migrationof the 18:2ω6 which was shown to be responsible for the majority ofanti-T cell activity. However, other fractions were also active,suggesting that several emu oil components might be responsible.

The data from the experimental section below have revealed avenues whichcould be used to standardise emu oil, particularly for itsanti-inflammatory activity. The results indicate that mice may be usedas models of testing systems for chronic (DTH) and acute (carrageenan)inflammatory diseases. These represent simple systems in whichinflammation can be readily quantified. To decrease variability, an iproute rather than topical emu oil administration is used. It has beenestablished that the efficacy of an emu oil preparation may bedetermined by establishing the extent to which the preparation can bediluted before anti-inflammatory activity is lost. In this system ofstandardisation, an established, active emu oil can be used as astandard against which other emu oils may be tested. A criterion foraccepting or rejecting emu oil preparations can then be established forthe industry. The standard can be based on the optimal renderingconditions, as well as storage of oils, feed for emus, breed of emu etc(Table 2). Oil prepared at 100° C. was found to have the highestanti-inflammatory activity, whilst oil prepared at 40° C. had minimalactivity.

Furthermore, the inventors found that the anti-inflammatory activity ofemu oil was strongest when administered after inflammation had occurred.Also, the inventors found that administration of the emu oil 1 h priorto inflammation has better anti-inflammatory efficacy than if the oil isadministered 3 h prior to inflammation. TABLE 2 PREPARATION OF EMU OIL(potential causes of variability) Collection of fat Age of animal DietGenetics Sex Length of time after death of the animal Storage conditionsof collected fat Lipase/phospholipase/lipoxygenase activity Non-enzymicoxidation Rendering Temperature of rendering Type of container usedAmount of water Surface area Length of rendering time FiltrationTemperature of filtration Type of filter Water in the filtrate Metalcontent Protein content Variable crystallisation Possible productsformed during the processing of emu fat Oxidation products of fattyacids Free fatty acids Lysophospholipids Conjugated linoleic acid Transisomers Diglycerides Monoglycerides Oxidation products of cholesterol

It is preferable to extend the testing by conducting in vitro assays tosupport the data from the in vivo chronic and acute inflammationreactions. This is particularly important before the oils can becommercially used. Thus, effect on T lymphocyte and monocyte functionfor chronic, and neutrophil function for acute, inflammation can beemployed. A model is illustrated in FIG. 2.

Both for the DTH and carrageenan inflammatory response, a relationshipcan then be established for the amount of oil versus the degree ofinhibition of inflammation.

From the graph of FIG. 1, the emu oil concentration required to achieve25% inhibition (ID₂₅) of the inflammatory responses can be deduced. Fromthis value, the anti-inflammatory power of the oil can be determined.The values may be computed for both acute and chronic inflammation,where they may be different.

The above anti-inflammatory efficacy values can be corroborated by datausing the ethanol soluble fraction of the oil, examining an effect on Tlymphocyte function and neutrophil function. Two useful parameters arelymphoproliferation for T lymphocytes and chemotaxis for neutrophils forchronic and acute inflammation respectively. Similar ID₂₅ and maximalinhibition values based on these parameters can be computed as discussedabove.

Based on the effects of emu oil on T lymphocyte and macrophageresponses, as well as neutrophil responses, the therapeutic potential isapparent for diseases/conditions summarised in Table 3. The targets inthe treatment of these inflammatory diseases are outlined, specificallythose which are critical and are targeted by emu oil. The targets of emuoil have been further expanded in FIG. 2, which shows the events whichlead to joint damage in rheumatoid arthritis. The T cells andmacrophages, as well as neutrophils, are targeted and either preventedfrom migrating into the tissue and/or prevented from being activated togenerate tissue destructive mediating cytokines. TABLE 3 Therapeutictarget for emu oil and the respective disease TARGETS RELEVANTCONDITION/DISEASE TO EMU OIL THERAPY Cardiovascular diseases Endothelialcells, macrophages Rheumatoid arthritis T cells, macrophages andneutrophils Atopic dermatitis T cells, interferon γ Inflammatory boweldisease T cells, macrophages, neutrophils Systemic lupus erythematosus Tcells and macrophages Asthma T cells, macrophages, neutrophils,cytokines Cystic fibrosis Macrophages and neutrophils Breastpain/premenstrual syndrome Oedema Transplantation T cells, cytokinesNeurological diseases T cells, macrophages Psoriasis T lymphocytes,interferon γ Diabetes renal, retinal and Endothelial cells, macrophages,cardiovascular complications neutrophils Gout Neutrophils Acuterespiratory distress syndrome Neutrophils, cytokines Acne Neutrophils,cytokines Septic arthritis Neutrophils, cytokines Reperfusion injuryNeutrophils, cytokines

In summary, the data herein has shown the complexity of the compositionof emu oil, in which the fatty acid content was studied in detail. Thereare no major differences in the levels of the various fatty acid speciesin distinctly different preparations, in terms of geography, feed,rendering and storage. Nevertheless, there is a marked difference in theability to depress inflammation. Using a freshly prepared standardisedemu oil preparation (Makin), the anti-inflammatory properties of emu oilwere tested, in chronic and acute in vivo and in vitro inflammationmodels. Some evidence points to at least some of the activity being dueto an unsaturated fatty acid, 18:2ω6, but the study has demonstrated thedifficulty in trying to identify what gives rise to theanti-inflammatory properties. Be that as it may, the inflammatory modelsdeveloped can be used to standardise the anti-inflammatory activity ofemu oil, which would seem to be a prerequisite for developing a viableindustry, using quality-controlled Australian oils.

Materials and Methods

Emu Oils

Details of the emu oils used in the study are outlined in Table 4. Theemu oils were kept frozen at −20° C. in aliquots. TABLE 4 Description ofthe different preparations of emu oils used in the present study Age ofbirds at Rendering Process Age of Oil slaughter Feed Makin Back fat @ 40C. 2 months 1-15 months old Feed lot mix G53 Gut fat @ 40 C. 4 years1-<3 years old Grainfed & range A2-100G Gut fat @ 40 C. 4 years 5-<3years old Grainfed & range Toowoomba Back fat @ 40 C. 4 years 2-<3 yearsold Grainfed & range Little Meadow Gut & back fat @ 2 years Unknown Emupellets & 104 C. range Gut Fat A Gut fat rendering 1 year 1-15 monthsold Farmed: Green temperature clovers, weeds & unknown grasses, milledBack Fat A Back fat rendering 1 year 1-15 months old barley, triticale,temperature wheat & luceme, unknown canola oil Gut Fat B Gut fatrendering 1 year 1-15 months old Back Fat B Back fat rendering 1 year1-15 months old temperature unknown Commercial Unknown Unknown UnknownUnknown1. Preparation of Ethanol Soluble/Insoluble Fractions

To obtain the ethanol soluble fraction, 2 ml of emu oil was mixed with 1ml of ethanol, centrifuged at 2,500 g/3 min/4° C. and the upper phasecollected. The extraction procedure was repeated three times on thelower phase. These ethanol soluble fractions were pooled, centrifugedand dried under N₂ gas stream. Eventually, stocks of 2 ml volume weremade for experiments; also, the ethanol insoluble fraction (EIF)remaining was retained as a rich source of triglyceride.

2. Fatty Acid Analyses

2.1 Thin Layer Chromatography

Up to 1 mg emu oil in 20-40 μl chloroform-methanol (4:1) was applied asa 1 cm band to the edge of a TLC plate. Linoleic acid (18:2) was appliedas a standard in a 0.5 cm band to one side of the test sample. Thechromatogram was developed in hexane-ether-acetic acid (80:20:1) anddried in the fume hood. The zones were viewed by exposure to 12 vapouror sprayed lightly with 18N H₂SO₄ and charred at 150° C. Larger amountsof Makin emu oil were dissolved in chloroform-methanol (4:1), andaliquots of the solution (equivalent to 5 mg of oil) were applied as a6-7 cm band to a silica thin layer plate. An equivalent amount of oliveoil dissolved in the same solvent mixture was applied to the plate as a6-7 cm band and served as a control. An unesterified fatty acid standardwas applied to the edges of the plate. A chromatogram was developed inhexane-ether-acetic acid (80:20:1) and, after drying, the plate wasexposed to iodine vapour.

2.2 NMR Analysis

This was performed by Dr N. Trout, Flinders University. To a dry flask(5 ml) was added 100-120 mg of the thawed emu oil (shaken thoroughly),which was dissolved in dry toluene (1-1.2 ml). To this was added afreshly prepared solution of sodium methoxide (75 mg Na in methanol (2ml)) under N₂. The resulting mixture was placed under reflux for ninetyminutes, before cooling and adding acetic acid (100 μl) and water (2.5ml). The white mixture was extracted with hexane twice before the layerswere dried over Na₂SO₄, filtered and the volatiles removed in vacuo. ¹³Cand ¹H NMR measurements were recorded on a Varian Gemini FT 300 MHzmultinuclear spectrometer, operating at 75.46 MHz and 300.75 MHzrespectively. All samples were dissolved in deuterated chloroform, usingthe central peak (77.0 ppm) for ¹³C and CHCl₃ (7.26 ppm) for ¹H NMRreferencing. To a NMR tube was added 75-100 mg of the emu oil followedby deuterated CDCl₃ (0.8 ml). The resulting solution was analysed byNMR. After one hour of pulsing, the spectrum was printed to show all thesignals indicative of a triglyceride.

2.3 GC Analyses

Child Health Research Institute (Dr. R. Gibson/Mr. M. Neumann). One dropof emu oil was methylated in 5 ml of 1% sulphuric acid (36N) in methanolfor 2 hours at 70° C. After cooling, the resulting methyl esters wereextracted into 2 ml of n-heptane and transferred to vials containinganhydrous sodium sulphate as the dehydrating agent. Emu oil fatty acidmethyl esters were separated and quantified using a Hewlett-Packard 6890gas chromatograph equipped with a 50 m capillary column (0.33 mm ID)coated with BPX-70 (0.25 μm film thickness—SGE Pty Ltd, Victoria,Australia). The injector temperature was set at 250° C. and the flameionisation detector at 300° C. The initial oven temperature was 140° C.and was programmed to rise to 220° C. at 5° C. per minute. Helium wasused as the carrier gas at a velocity of 35 cm per second. Fatty acidmethyl esters were identified based on retention time to authentic lipidstandards from Nuchek Prep Inc (Elysian, Minn.).

RMIT (Prof A. Sinclair/Ms. K. Murphy): Samples were analysed induplicate. An aliquot of whole lipid was taken and dried using a streamof nitrogen. Samples were hydrolysed to free fatty acids using 7.9% KOH(Univar, AJAX chemicals, Australia) in methanol (Merck, Germany).Samples were cooled and converted to fatty acid methyl esters (FAME)using 20% boron trifluoride (BF₃) in methanol complex (Merck, Germany).Gas Chromatographic analyses were performed using a Shimadzu GC 17A GCfitted with a flame ionisation detector (FID). FAME were analysed usinga BPX-70 50 m cross-linked 70% Cyanopropyl Polysilphenylene-siloxanecapillary column with an ID of 0.32 mm and 0.25 μm film thickness.Samples were injected at 125° C. and held for 1.0 minute. The oventemperature was set to increase by 5° C./min to 170° C. and held for 4minutes, then by 0.5° C./min to 175° C. and 4° C./min to a finaltemperature of 220° C. which was held for 3 minutes. The injector anddetectors were maintained at 260° C. and helium was used as the carriergas. Peak area and concentrations were quantified on an IBM compatiblecomputer using Shimadzu software (Japan).

2.4 GC-MS

GC-MS analysis was performed on a Varian Saturn 4D instrument with a J&WDB 5% phenylmethylpolysiloxane column (30 m×0.25 mm id).

2.5 MS

Women's and Children's Hospital (Dr. D. Johnson): 1 mg of emu oil wastreated with benzene/methanol/acetyl chloride at 100° C. for 90 min.After cooling, the neutralised solution was extracted with hexane andsamples of the extract were injected into a Perkin Elmer Turbomass MassSpectrometer.

3. Sterol Analysis

These experiments were carried out by Ms K Murphy from the laboratory ofProfessor A. Sinclair at the Royal Melbourne Institute of Technology.Sterol-enriched fractions were obtained from two emu oil samples (Makinand G53) by alkaline saponification with 5% KOH in methanol/water(80:20, v/v), followed by extraction with 2 ml of hexane:chloroform(4:1, v/v) three times. The sterols were then converted to theircorresponding trimethylsilyl ethers (OTMSi) with BSTFA(N,O-Bis(trimethylsilyl)trifluoroacetamide) for 15 minutes at 70° C. Gaschromatographic analyses were performed using a Shimadzu GC 17A GCfitted with a FID and a BPX-5 50 m (5% Phenyl Polysilphenylene-siloxane)with an ID of 0.32 mm and 0.25 μm film thickness. Samples were injectedat 200° C. and held for 1 minute. The oven temperature was set toincrease by 20° C./min to 340° C. and held for 30 minutes. The injectorand detector were maintained at 280° C. and helium was the carrier gas.Peak area and concentrations were quantified on an IBM compatiblecomputer using Shimadzu software (Japan).

4. Analysis of Phenolics

The analysis of phenolics in a sample of Makin emu oil, in two other emuoils, and in a number of other fats and oils was carried out in thelaboratory of Dr P. Hayball at the University of South Australia. Thetotal phenolic content was determined using a modification of theFolin-Ciocalteau method and results were expressed as gallic acidequivalents.

5. Inflammation Models

5.1 Delayed type hypersensitivity (DTH) reaction: The DTH response wasinduced in 12 week old female BALB/c mice (Animal Resource Centre,Perth) as described previously (8). Briefly, mice were injected withsheep red blood cells (100 μl of 10% haematocrit) (SRBC; Sigma ChemicalCo.). After 5 days, the animals were challenged intradermally in theright hind footpad with SRBC (25 μl of 40%-haematocrit) or into the leftfootpad with diluent (25 μl). The DTH response was determined 24 h postchallenge and was calculated by comparing the thickness between thediluent vs SRBC injected footpads. Footpad thickness was measured with adial calliper.

5.2 Carrageenan-induced paw reaction: Carrageenan-induced paw reactionwas induced as described previously (9,10). Mice were inoculated withcarrageenan (1 ml/kg of a 1% solution) (Type IV; Sigma Chemical Co.)into the right hind paw. The reaction was assessed by measuring hind pawthickness at the indicated times.

6. Leukocyte Separation

Mononuclear leukocytes (MNL) and neutrophils were prepared by the rapidsingle-step separation method (11). Briefly, whole blood was layeredonto Hypaque-Ficoll medium of density 1.114 and then centrifuged at 400g/30 min. After centrifugation, the leukocytes resolve into two distinctbands. The upper band contained MNL and the lower band the neutrophils.

7. Lymphocyte Proliferation

Lymphocyte proliferation was measured by a semi-automated microtechnique(12). Human mononuclear cells (2×10⁵) were seeded into u-bottomed wellsof a micro-titre plate (50 μl) and treated with 500 of the ethanol emuoil fraction. After 30 min incubation, 2 μg/μl PHA was added tostimulate the T lymphocytes. The cells were incubated for 72 h at 37° C.in an atmosphere of 5% CO₂-air and high humidity. At 6 h prior toharvest, the cultures were pulsed with 1 μCi of ³H-TdR. The cells wereharvested and the amount of radioactivity incorporated measured in aliquid scintillation counter.

8. Cytokine Production

Production of IL-2, IFN-γ and lymphotoxin (TNF β) by T lymphocytes wasmeasured in MNL stimulated with PHA as described for lymphocyteproliferation. The supernatants from cell cultures were collected andthe amount of cytokine measured by ELISA using cytokine specificmonoclonal antibodies as described previously (13).

Production of the cytokine TNF α by monocytes was measured in MNLstimulated with LPS. Briefly, 2×10⁵ MNL in a 100 μl volume was added toflat bottomed wells of a microtitre plate and then the cells werestimulated by adding 100 μl of 200 ng/ml bacterial lipopolysaccharide(LPS). After incubation at 37° C./48 h, the supernatant was collectedfor TNF a measurement, using an ELISA and TNF a specific monoclonalantibody as described previously (13).

9. Neutrophil Adhesion

9.1 To Plasma Coated Surfaces

Adhesion was assessed by the ability of neutrophils treated with emu oilextract to bind to plasma-coated plates after stimulation with TNF α.Plates which had been coated with autologous plasma (1:10), washed anddried received 50 μl neutrophils (5×10⁶/ml) which were treated for 30mins at 37° C./5% CO₂. The neutrophils were stimulated with TNF α (10³units/ml) for 30 mins at 37° C./5% CO₂, washed with HBSS, then stainedwith 100 μl Rose Bengal (0.25% w/v PBS) at room temperature.Non-adherent cells were removed by washing with HBSS, and then 200 μlethanol: PBS (1:1) was added and development proceeded at roomtemperature for 30 mins before reading on a plate reader at 570 nm.

9.2 Neutrophil Adherence to Human Umbilical Vein Endothelial Cells(HUVEC).

HUVECs were isolated from umbilical cords stored at 4° C. afterdelivery, as previously described (15) but with 0.2% (w/v) gelatin(Cytosystems) to coat all tissue culture flasks and plates, 0.07% (w/v)collagenase (from Clostridium histolyticum, type II, Worthington) todigest the interior of the umbilical vein, and a culture mediumconsisting of RPM1640 (ICN-Flow) containing 40 mmol/l TES, 15 mmol/lD-glucose, 80 U/ml penicillin (Flow), 80 μg/ml streptomycin (Flow), and3.2 mmol/l L-glutamine, which was brought to 260 to 300 mOsm/l beforethe addition of 20% (v/v) pooled, heat-inactivated (56° C., 30 minutes)human group AB serum. Endothelial cells were identified by theircharacteristic contact-inhibited cobblestone morphology and positivestaining for factor VIII-related antigen using peroxidase-conjugatedanti-rabbit IgG to human von Willebrand factor (Dako) and3,3′-diaminobenzidine.

Confluent cultures were subcultured after 2 to 5 minutes exposure totrypsin (0.05% [v/v], Flow)-EDTA (0.02% [w/v]). For experimental use,second-passage cells were plated at 2×10⁶ cells per well per 0.2 mlculture medium in 96-well culture plates. The HUVECs were treated withthe emu oil ethanol soluble fraction and then with TNF-α, the monolayerswere washed once with RPMI 1640, before incubation for 30 minutes at 37°C. in the absence or presence of 5×10⁵ neutrophils in E-SFM (finalvolume, 100 μl). Nonadherent cells were removed by gentle aspiration,and the wells were washed twice with HBSS containing 0.1% (w/v) μMphorbol myristate acetate (PMA) to stimulate the cells' BSA beforestaining with rose bengal. After release of the dye with 50% ethanol,the absorbance (570 nm) of each well was determined with an ELISA platereader. Test and blank wells were performed in triplicate. Results werecalculated after subtraction of the mean blank value (withoutleukocytes) from each test value (plus leukocytes) (15).

10. Neutrophil Chemotaxis

Chemotaxis was measured by the migration under agarose method aspreviously described (16). Six millilitres of 1% molten agarose inmedium 199 containing 5% fetal calf serum were poured into petri dishes.After the agarose solidified, sets of three holes/wells were punched inthe agarose layer. Plates with these sets of three wells were used tomeasure leukocyte migration in a chemotaxis gradient, with 50 μl of1×10⁻⁷M fMLP, 5 μl of neutrophils (2.5×10⁵) and 5 μl of medium 199 beingadded to the inner, centre and outer wells respectively. Two well setswere used to measure random migration, cells being added to one well andmedium to the other. The plates were incubated at 37° C. and thedistance of cell migration measured directly under a phase-contrastmicroscope after 90 min. The approximate migration distances ofneutrophils in assays conducted in our laboratory were 2.2 mm and 0.7 mmin the presence and absence of fMLP, respectively.

11. Results

11.1 Chemical Composition of Emu Oil

Analyses of emu oil were conducted at a number of different centres toenable a better assessment of the various constituents of the oil. Fattyacid analyses of emu oils were made at the Women's and Children'sHospital in Adelaide, Flinders University, and at the Royal MelbourneInstitute of Technology (RMIT), Victoria. Analysis of phenolic contentof the oil was conducted at the University of South Australia and sterolanalysis at RMIT. The results are all presented and, in some cases,comparisons between the same oils from analyses made at differentcentres are outlined.

11.2 Fatty Acid Composition of Emu Oils

Examination by thin layer chromatographic analysis of emu oil showedthat the major component of emu oil is triacylglycerol. However, smalleramounts (around 1-2%) of at least 7 other minor components were detected(FIG. 3). Three of these were tentatively identified as unesterifiedfatty acids, diacylglycerol, and sterols.

The identity of the other components was not established. Some of thesehad a similar chromatographic mobility to compounds present in oliveoil. These experiments indicate that emu oil is a more complex mixturethan previously believed. As many of the minor components in olive oilare thought to contribute to its properties, particularly its healthbenefits, it is likely that the minor components in emu oil may alsohave a similar effect. Apart from a band in olive oil running near thesolvent and tentatively identified as the hydrocarbon, squalene, thechromotagraphic profile of emu oil did not appear very different fromolive oil, although it is likely that there are some components that areunique to each oil.

The fatty acid composition of the nine emu oils analysed by GC-MS atFlinders University by Dr Neil Trout (organic chemist) is shown in Table5. The predominant fatty acid was oleic acid (18:1ω9). This ranged from49% to 58% of the fatty acids in the nine oils. The next most prominentfatty acid was palmitic acid (16:0), which ranged from 19-22%. Otherprominent fatty acids were stearic acid (18:0) ranging from 9-11%,linoleic acid (18:2ω6) ranging from 5.5-17% and hexadecenoic acid(16:1ω7) ranging from 3-6%. A typical GC-MS trace of the fatty acidanalyses is seen in FIG. 4. TABLE 5 GC-MS Analysis of nine preparationsof emu oil. GC-MS analyses were performed on a Varian Saturn 4Dinstrument with a J&W DB5/phenylmethyl polysiloxane column (30 m × 0.25mm). fatty acid Emu oil 14:0 14:1 16:0 16:1 17:0 18:0 18:1 18:220:0/20:1 Little Meadow Trace trace 20.18 5.79 trace 8.84 50.12 10.40trace  4.65 trace trace Toowoomba Trace trace 20.17 3.63 trace 11.6049.12 9.04 trace  3.23 trace trace Gut Fat A Trace trace 21.35 5.22trace 10.45 48.87 9.21 trace  4.89 trace trace G53 Trace trace 20.133.88 trace 11.65 58.33 2.79 trace  2.70 trace trace A2-100G Trace trace19.48 3.98 trace 11.64 54.28 5.45 trace  4.60 trace trace Makin Tracetrace 18.92 3.53 trace 11.04 49.60 14 trace  2.91 trace trace Back Fat ATrace trace 22.25 5.27 trace 10.92 49.31 8.38 trace  3.86 trace traceDuncan 170M Trace trace 19.65 3.50 trace 10.13 52.32 11.13 trace  3.26trace trace Duncan 176M trace trace 19.20 2.85 trace 8.83 49.78 16.70trace  2.70 trace trace

Analyses of these oils were also undertaken in Dr Bob Gibson'slaboratory at Flinders University (Table 6). Nine emu oil samples wereanalysed by this method. Examination of GC traces showed that the fattyacid composition was much more complex than had been suspected, withupwards of two dozen different fatty acids identified. Many of thesecomponents were only present in trace amounts (<0.1%). Emu oil containsmainly straight chain even numbered carbon chain fatty acids, the majorsaturates being palmitic (16:0) and stearic (18:0) acids, with onlysmall amounts of shorter (14:0) and longer (20:0 and 22:0) chainsaturates (Table 6). TABLE 6 GC Analyses of emu oil fatty acids FattyAcid Gut FatB GutFatA A2-100G 53G Back FatA Back FatB Little MeadowToowomba Makin  8:0  9:0 10:0 11:0 12:0 0.03 0.03 0.03 0.03 0.03 0.020.03 0.04 0.03 13:0 14:0 0.30 0.34 0.25 0.28 0.33 0.30 0.33 0.26 0.4215:0 0.03 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.07 dma 16:0 16:0 23.8823.18 20.00 19.27 23.91 23.80 23.71 20.36 20.54 17:0 0.09 0.10 0.10 0.140.10 0.09 0.09 0.12 0.22 dma 18:0 18:0 10.85 8.47 9.42 11.52 8.75 9.948.28 10.73 11.14 20:0 0.19 0.21 0.16 0.18 0.18 0.18 0.12 0.17 0.21 22:00.03 0.02 0.02 0.03 0.02 0.03 24:0 Total Sats 35.40 32.38 30.01 31.4733.35 34.39 32.58 31.71 32.67 Trans 16:1 0.23 0.07 0.03 Trans 18:1ω90.24 0.24 0.30 0.39 0.05 0.24 0.91 0.37 0.32 Trans 18:1ω7 0.06 0.19Trans 18:2 0.01 0.02 0.04 Totals Trans 0.29 0.26 0.30 0.39 0.28 0.241.00 0.37 0.58 11:1 12:1 13:1 14:1 0.07 0.11 0.07 0.07 0.11 0.09 0.110.06 0.09 15:1 16:1ω9 0.10 0.11 0.13 0.12 0.10 0.10 0.18 0.13 0.1516:1ω7 3.94 4.97 3.57 3.08 5.23 4.58 5.33 3.22 2.95 17:1 18:1ω9 48.1748.82 52.99 49.71 47.94 48.42 47.48 49.57 47.88 18:1ω7 2.32 2.60 2.392.15 2.55 2.27 2.72 2.05 2.68 19:1 0.02 0.02 0.05 20:1ω11 0.05 0.05 0.090.06 0.05 0.05 0.06 0.08 0.06 20:1ω9 0.47 0.45 0.40 0.48 0.46 0.45 0.290.45 0.41 22:1ω11 22:1ω9 0.07 0.05 0.02 0.04 0.06 0.06 0.03 0.04 0.0224:1ω9 Total Monos 55.19 57.18 59.65 55.70 56.50 56.02 58.18 55.56 53.4118:2ω9 0.04 0.04 0.02 0.04 0.04 0.04 0.02 20:2ω9 0.03 0.05 0.03 20:3ω90.03 0.03 0.04 0.03 Totalω9 48.84 49.47 53.61 50.35 48.60 49.08 48.0450.27 49.26 Totalω7 6.26 7.57 5.95 5.21 7.78 6.85 8.05 5.27 4.78 9.1118:2 cLA 0.05 0.01 0.05 0.05 0.05 0.05 0.03 0.04 0.06 10.12 18:12 cLA18:2ω6 7.81 8.65 9.26 11.26 8.32 7.92 9.18 11.59 11.95 18:3ω6 0.04 0.040.02 0.04 0.04 0.02 0.04 20:2ω6 0.05 0.05 0.07 0.10 0.06 0.05 0.05 0.080.10 20:3ω6 0.02 20:4ω6 0.03 0.04 0.06 0.06 0.04 0.03 0.04 0.06 0.0922:2ω6 0.00 22:4ω6 0.03 0.03 0.04 22:5ω6 Totalω6 7.92 8.79 9.44 11.428.47 8.05 9.30 11.76 12.24 16:2ω3 0.02 0.03 0.03 0.02 0.03 0.08 18:3ω31.12 1.29 0.41 0.91 1.26 1.18 0.80 0.43 0.82 18:4ω3 20:3ω3 20:5ω3 22:5ω30.02 0.02 0.04 0.03 0.03 0.04 22:6ω3 Totalω3 1.12 1.34 0.46 0.98 1.311.21 0.82 0.43 0.94

The ranges for the predominant fatty acids were 18:1ω9 (47-53%), 16:0(20-24%), 18:0 (8-11%), 18:2ω6 (8-12%) and 16:1ω7 (3-5%). Again, thegreatest variability was seen in 18:2ω6 and 18:0. The main monoenoicacid was oleic acid (18:1ω9). Traces of shorter (16:1ω9) and longerchain (20:1ω9, 22:1ω9) monoenoic acids were detected. ω7 seriesmonoenoic fatty acids were also present, the main one being 16:1ω7,which was present in significant amounts (around 3%). Only traces of oddnumbered carbon chain fatty acids were detected. The mainpolyunsaturated fatty acid was linoleic acid (18:2ω6). Traces of otherω6 series polyunsaturated fatty acids were present, and included gammalinolenic (18:3ω6), arachidonic (20:4ω6), and docosatetraenoic (22:4ω)acids. ω3 Polyunsaturated fatty acids were minor components, the mainone being alpha linolenic acid (18:3ω3), with only traces of 16, 20, and22 carbon compounds. Conjugated linoleic acid (the 9, 11 isomer) wasalso detected, but only in very small amounts (<0.1%).

Fatty acid analysis was also carried out by Ms K. Murphy in thelaboratory of Professor Andrew Sinclair at RMIT. Approximately 21individual fatty acids were identified in the emu oils (Table 7). Thedominant fatty acid class was the monounsaturated fatty acids(approximately 54-57%), followed by the saturated fatty acids (31-34%).Omega-6 fatty acids were the dominant polyunsaturated fatty acidsidentified, ranging from 8-12%, while omega-3 fatty acids were presentat less than 2% of total PUFA.

Oleic acid (18:10ω9) was the dominant fatty acid in the emu oils (Table7), ranging from 48.2% in the G53 emu oil to 49.2% in the Makin emu oil.Palmitic acid (16:0) was the next most dominant fatty acid(approximately 19-23%), followed by stearic acid (18:0) (10-11%),linoleic acid (18:20ω6) (8-12%) and hexadecenoic acid (cis16:1ω7)(3-4%). DHA predominated in the tuna oil, followed by 16:0, 18:1 ω9,18:0, EPA, and cis 16:1ω7. Olive oil was predominantly 18:1ω9 (78%),with a smaller percentage of 16:0 (11%) and 18:0 (3%). TABLE 7 GCAnalysis of fatty acids of emu, tuna and olive oils at RMIT Oil Fattyacid Emu (Makin) Emu (53G) Emu (KM) Tuna Olive 12:0 0 0.42 0.14 0.01 012:1 0 0 0 0.70 0 14:0 0.49 0.29 0.14 2.59 0 14:1 0.06 0.43 0 0.85 015:0 0 0.01 0 0.77 0 16:0 21.94 18.82 23.33 17.07 11.09 16:1ω7t 0.120.15 0 0.28 0.05 16:1ω7c 3.13 3.00 3.61 3.31 0.66 17:0 0.12 0.22 0.041.85 0.08 17:1 0.06 0.03 0 0.86 0.03 18:0 11.32 11.0 9.53 6.27 2.8718:1ω9 49.2 48.24 51.32 13.04 77.84 18:1ω7 1.77 2.09 2.42 2.17 1.6618:2ω6 11.90 10.31 8.18 1.88 5.88 18:3ω3 0.75 0.89 1.61 0.54 0.16 18:4ω30 0 0 0.87 0 20:0 0.03 0.22 0.01 0 0.02 20:1ω11 0.07 0.53 0.07 0.60 0.0120:1ω9 0 0 0 1.55 0 20:1ω7 0 0 0 0.12 0 20:2ω6 0.01 0.55 0 0.31 0 20:4ω60 0.17 0 2.55 0 20:3ω3 0 0 0 0.12 0 20:4ω3 0 0 0 0.60 0 20:5ω3 0 0 06.01 0 22:4ω6 0 0.62 0 1.06 0 22:5ω6 0 0 0 0.4 0 24:0 0 0.01 0 1.72 022:5ω3 0 0 0 1.14 0 22:6ω3 0 0 0 23.46 0All figures are percent of total fatty acids present in the oil.

Examination of the GC/Mass spectrometric analysis of emu oil fatty acidsby Dr. D. Johnson at the Women's and Children's Hospital confirmed thatthe main fatty acid components of emu oil were 14:0, 16:1, 16:0, 18:1,18:2, 18:0, 20:0 and 20:1 (see FIG. 4). However, two other components,labelled as peaks 1 and 2, were also detected. These were not present inanalyses carried out by two other laboratories. Neither peak waspositively identified as a fatty acid, even though the 74 mass ion,indicative of fatty acid esters, was detected in both and wasparticularly prominent in peak 2. Based on a comparison of the peakheights as compared to other fatty acid peaks, peak 2 constituted around34% of the total fatty acids in one of the emu oil samples analysed(Makin) and 6-7% in the other (A2-100G).

To explore the possibility that these two components were hydroxy fattyacids, samples of emu oil were hydrolysed with benzene/methanol/1%sulphuric acid at 100° C. for 2 hours. After extraction into hexane,samples of the hydrolysate were chromatographed on a TLC plate inhexane-ether-acetic acid (80:20:1) and the zones were detected byexposure to iodine vapour. Although under these conditions there hadbeen almost complete hydrolysis of the emu oil, there was no evidencefor the presence of hydroxy fatty acids. The only components detectedwere normal (unhydroxylated) fatty acid esters together with smallamounts of alkali-stable lipids. One other possibility is that peaks 1and 2 were formed by acetylation of diacylglycerols. Most animal andplant fats, including emu oil, contain small amounts of diacylglycerolgenerally formed by the breakdown of triacylglycerols. This possibilityhas not been investigated further.

11.3 Sterol Analysis

Approximately thirty sterols were present in the emu oils and tuna oil,while 28 sterols were present in the olive oil (Table 8). Of those, 15sterols of the emu oils and 19 of the olive oil could not be identifiedwith gas chromatography not linked to a mass spectrometer. Data has beenpresented as percentage of total sterols. Cholesterol was the majorcomponent of the sterol fraction of both Adelaide emu oil samples. Itcomprised 70% of the Makin and 55% of the G53 emu oil sterolsrespectively. A further 14 sterols were identified. The only othercomponent present in significant amounts was 4, 23,24-trimethyl-5α-cholest-22E-en-3β-ol (3.7 and 7.1%). TABLE 8 Sterolanalyses of emu, tuna and olive oil Sample Oil (% of total sterols)Sterol Emu (Makin) Emu (53G) Emu KM Tuna oil Olive oil Totalunidentified peaks 18 33 34 10 64 5α-cholestane 0.5 0.3 0.2 0.5 1.124-nordehydrocholesterol 1 0.5 0.1 3.1 1.0 C26 sterol 0.6 0.1 2.3 0 3.2Patinosterol 0.6 0.1 0.1 0 1.1 Trans-22-dehydrocholesterol 0.6 0.1 0 0.90 Cholesterol 70 55 43 85 5 Cholestanol 1.5 1.1 0.3 0 0 Desmosterol 0.90.6 1.2 0 0 Brassicasterol 0.9 0.1 1.7 0 0 24-methylenecholesterol 0.1 02.9 0 0.8 24-methycholesterol 0.2 0.2 1.5 0 1.5 Stigmasterol 0.9 0.7 1.90 0 β-sitosterol 0.7 1.3 0.8 0 21 Isofucosterol 0.2 0.6 0 0.1 04,23,24-Trimethyl-5α-cholest- 3.7 7.1 10.0 0 1 22E-en-3β-olAll figures are percent of total sterol present in the oil.

A number of the other components, such as sitosterol, brassicasterol,and sitosterol, are plant sterols and therefore probably derived fromthe diet. A further 15 components, many of which are believed to besterols, were also detected but they were not identified. These dataprovide further evidence for the complexity of emu oil and for thevariability of its composition. The presence of plant sterols indicatesthat the concentration and composition of the minor components may beaffected by diet.

Other sterols present in the Makin and G53 emu oils were an unidentified(UI) sterol eluting before cholesterol (5 and 13% respectively), an UIsterol eluting before 4,23,24-trimethyl-5α-cholest-22E-en-3β-ol (5 and5% respectively), and cholestanol (2 and 1% respectively). Theunidentified peaks were present in all samples tested and cannot beidentified until gas chromatography with mass spectrometry is applied.

There were also traces of several additional sterols, including5∝-cholestane, 24-nordehydrocholesterol, C26 sterol, patinosterol,trans-22-dehydrocholesterol, desmosterol, brassicasterol,24-methylenecholesterol, 24-methylcholesterol, stigmasterol,β-sitosterol and isofucosterol (all ≦1%). β-sitosterol was the majorsterol in the olive oil sample (21%). The peak identified as cholesterol(5%) in the olive oil sample is unlikely to be cholesterol. There is apossibility that it could be a long chain alcohol (28:0) which runs veryclose to cholesterol.

Tuna oil was comprised mainly of cholesterol (85%).

11.4 Polyphenol Analysis

The highest concentration of phenolics was found in olive oil, withvalues as high as 708 μmoles per litre (Table 9). Levels were very lowin a number of other plant oils (sunflower, canola, and soya bean oils).The Makin emu oil had levels of phenolics that were comparable to thosedetected in castor and peanut oils (25.0 vs 21.7 and 25.0 and 27.1 and30.0 mol per litre) (Table 9). As phenolics are normally found inplants, it is likely that the emu oil phenolics are derived from dietarysources. The total phenolic fraction of olive oil and other dietary oilsnormally comprises a mixture of simple and complex phenols. Although theemu oil phenolics were not identified, it is likely that they include amixture of compounds. Their presence is a further indication of thecomplexity of emu oil. In view of their powerful antioxidant properties,and their ability to modulate the activity of immune cells (17), it ispossible that they contribute to anti-inflammatory activity of emu oil,either directly or synergistically with other components present in theoil. TABLE 9 Phenolic content in a range of plant and animal oils/fats.SAMPLE Phenol concentration (μmol/l) Canola Oil (No Frills) 0.0 LiquidParaffin BP 0.0 Water 0.0 Sunflower Oil (Sunbeam) 1.4 Canola Oil (NoFrills) 1.4 Water 1.7 Sunflower Oil (Sunbeam) 5.7 Liquid Paraffin BP 8.3Soya Bean Oil 8.6 Soya Bean Oil 8.6 Ghee 15.7 Emu Oil (Emu Fire) 13.3Ghee 15.7 Emu Oil (Emu Fire) 18.3 Castor Oil BP 21.7 Castor Oil BP 25.0Peanut Oil 27.1 Peanut Oil 30.0 Olive Oil (J. Laforgia, Young Trees2000) 690.0 Olive Oil (J. Laforgia, Young Trees 2000) 708.6 Makin EmuOil 25.012. Anti-Inflammatory Properties of Emu Oil12.1 The Effect of Emu Oil on the Chronic Inflammatory Reaction

In these experiments, the Makin emu oil preparation was primarily used,as this had been prepared under “guided” conditions. The chronicinflammatory response was measured by the delayed type hypersensitivityreaction. This reaction is initiated by an antigen and elicitedfollowing antigen challenge at various sites. The response ischaracteristic of sensitised T lymphocytes, which mobilize andaccumulate at the antigen challenge site. Such cells then cause thenon-specific accumulation of other lymphocytes and a large infiltrationof macrophages. This represents a significant model of the reactionsseen in inflammatory diseases where tissue damage occurs. In theseinvestigations, we used sheep red blood cells (SRBC) as the antigen forthe delayed type hypersensitivity response. Mice were primed with SRBCsubcutaneously and after 5 days challenged in the footpad with SRBC andthe amount of swelling measured 24 h later. In these investigations, theeffects of emu oil on the inflammatory response were evaluated byinjecting 50 μl of the Makin emu oil intraperitoneally, three hoursprior to the antigen challenge. The data presented in FIG. 5 show thatmice which had been pretreated with emu oil developed a significantlydepressed DTH response, thus showing that emu oil has anti-inflammatoryactivity.

This activity of emu oil was found to be proportionately decreased asthe amount of emu oil injected was decreased (FIG. 6). Thus, when 120 μlwas injected, there was approximately 70% suppression of the DTHresponse, compared to 25% with 30 μl emu oil.

Several experiments were conducted to examine the reproducibility of theeffects of Makin emu oil on DTH inflammation. The oil was administeredin 50 μl ip. The results presented in Table 10 show that, in all cases,the emu oil was active in suppressing the inflammatory response. TABLE10 Summary of experiments examining the effects of Makin emu oil on theDTH response Experimental % inhibition of DTH response Number (Mean ±sem) 1 42.9 2 46.7 3 38.8 4 43.5 5 25.2 6 56.0 Mean ± sem 42.2 ± 4.1Mice were immunised subcutaneously with SRBC and 5 days later challengedwith SRBC subcutaneously in the hind footpad. Three hours prior tochallenge, the mice were treated with 50 μl of emu oil ip. The DTHreaction was assessed by measuring the thickness of footpad swelling.Five mice per group were used in each experiment.

A commercial source of emu oil cream from Emu Oil Therapies (EOT)designated as C1 was tested. The ointment is for topical application andcontains small amounts of eucalyptus and lavender oils. The cream wasapplied to the footpads of mice 1 h prior to challenge with SRBC. Theresults presented in FIG. 7 show that C1 was highly immunosuppressive,causing a 60% reduction in footpad swelling.

12.2 Comparison of the Anti-Inflammatory Properties of Different Emu OilPreparations

The various emu oil preparations which had undergone chemical analyseswere also compared in their ability to reduce the inflammatory response.Groups of mice were sensitised with SRBC and, 3 h prior to antigenchallenge, received one type of emu oil intraperitoneally. It is evidentfrom the results presented in FIG. 8 that Makin emu oil was the mosteffective. The others showed very poor anti-inflammatory activity.

12.3 Comparison of the Pre and Post Antigen Challenge Treatment with EmuOil

The utility of a substance to treat an inflammatory reaction can beassessed on its ability to stop inflammation even after it has beenelicited. This was examined for emu oil using the DTH model. In initialstudies, experiments were conducted in which the emu oil pretreatmenttime was varied from 1 to 5 h prior to challenge. Thus, SRBC primed micewere pretreated at 1,3 and 5 h prior to SRBC challenge with 50 μl ofMakin emu oil intraperitoneally. The results showed that the oil wasmost effective if given 1 h prior to challenge (FIG. 9).

In further experiments, the effects of delaying treatment of mice withemu oil until 3 h after challenge with SRBC on the development of theDTH reactions were examined. Investigations were set up to compare theeffects of 3 h pre-treatment versus 3 h post-treatment in relation toantigen challenge. The results showed that Makin emu oil was just aseffective if the treatment were delayed and, in fact, delayed treatmentwas significantly more suppressive than treatment given prior tochallenge (FIG. 10).

12.4 Effects of Emu Oil on Acute Inflammation

Acute inflammation is dominated by neutrophils rather than T lymphocytesand macrophages, although the latter two cell types are also likely tohave a role. This can be tested using an established model ofcarrageenan induced inflammatory responses. This model was used toexamine the effects of emu oil on acute inflammation. Mice were treatedintraperitoneally with Makin emu oil 3 h prior to receiving carrageenaninto the hind footpad. The swelling was then measured 24 h after theinjection of carrageenan. The data showed that the oil was quiteeffective in depressing the carrageenan-induced inflammatory response(FIG. 11). As per DTH reaction, comparison of pretreatment of mice for 1h, 3 h, 5 h showed 1 h to be most effective (FIG. 12).

Examination of emu oil post-treatment with respect to acute inflammationand carrageenan-induced inflammation showed that the delayed treatmentwas just as effective with this model in inhibiting inflammation (FIG.13). As with chronic inflammation, a greater degree of suppression ofinflammation was seen.

12.5 Effect of Rendering Temperature on Emu Oil Chemical Composition andAnti-Inflammatory Activity

Makin emu fat (EF) was subjected to heating at 40° C. for 2 h, the oilremoved and the remaining fat subjected to heating at 60° C. for 2 h.After collection of the oil, the fat was heated at 80° C. and the oilproduced under this temperature collected.

The oils prepared under the three different rendering conditions wereanalysed by GC. The results are presented in Table 11. TABLE 11 GCAnalyses of fatty acids in emu oils prepared at different renderingtemperatures Fatty Acid makin EO1-40C. EO1-60C. EO1-80C.  8:0  9:0 10:011:0 12:0 0.03 0.03 0.30 0.03 13:0 14:0 0.42 0.39 0.37 0.40 15:0 0.070.03 0.03 0.03 dma 16:0 16:0 20.54 26.65 26.71 27.13 17:0 0.22 0.08 0.090.09 dma 18:0 18:0 11.14 8.12 8.49 7.90 20:0 0.21 0.10 0.10 0.09 22:00.03 24:0 Total Sets 32.67 35.39 35.82 35.87 Trans 16:1 0.03 Trans18:1ω9 0.32 0.23 0.23 0.23 Trans 18:1ω7 0.19 Trans 18:2 0.04 TotalsTrans 0.68 0.23 0.23 0.23 11:1 12:1 13:1 14:1 0.09 0.14 0.13 0.16 15:116:1ω9 0.15 0.10 0.10 0.11 16:1ω7 2.95 5.71 5.51 6.05 17:1 18:1ω9 47.8846.37 48.31 48.64 18:1ω7 2.68 2.71 2.72 1.84 19:1 0.05 20:1ω11 0.06 0.060.06 0.06 20:1ω9 0.41 0.25 0.25 0.23 22:1ω11 22:1ω9 0.02 24:1ω9 TotalMonos 53.41 55.32 55.06 54.90 18:2ω9 0.02 20:2ω9 0.03 20:3ω9 0.03 0.020.03 0.03 Total ω9 49.26 46.75 48.89 46.00 Total ω7 4.78 8.42 8.22 8.739,11 18:2 cLA 0.06 0.05 0.05 0.05 10,12 18:2 cLA 18:2ω6 11.95 8.19 7.988.30 18:3ω6 0.04 0.02 20:2ω6 0.10 0.06 0.06 0.06 20:3ω6 0.02 20:4ω6 0.090.04 0.04 0.05 22:2ω6 22:4ω6 0.04 22:5ω6 Total ω6 12.24 8.28 8.10 8.4116:2ω3 0.08 0.02 18:3ω3 0.82 0.67 0.65 0.70 18:4ω3 20:3ω3 20:5ω3 22:5ω30.04 0.02 22:6ω3 Total ω3 0.94 0.67 0.89 0.70

The results showed that the three preparations were almost identical interms of composition of the major and minor fatty acids. When comparedto other emu oil preparations, the composition of fat was similar.

The three oils were then tested for their effects on thecarrageenan-induced inflammatory response. Mice were pretreated for 3 hand 120 μl of each of the emu oil preparations (40° C., 60° C. or 80°C.) and then treated with carrageenan in the hind paw. The result showedthat, while all three inhibited the inflammatory reaction, 60° C.rendering produced the most effective oil followed by 80° C. (FIG. 14).While the rendering temperature effects were also seen in the DTHreaction, it was the 80° C. and 100° C. oil preparations which were mostanti-inflammatory (FIG. 15).

12.6 Activity of the Ethanol Soluble Fraction of Emu Oil

The ethanol soluble component of Makin emu oil was prepared and examinedfor anti-inflammatory properties by using several in vitro parameters ofinflammation. The ethanol soluble fraction was tested for ability todepress T lymphocyte, macrophage and neutrophil responses.

12.6.1 T Lymphocyte Responses

Makin emu oil was subjected to solubility in ethanol. This ethanolsoluble oil fraction was then tested for ability to depressproliferation of mitogen stimulated human lymphocytes. The mononuclearcells were isolated from peripheral blood and pretreated for 30 min withdilutions of the fraction and then challenged with phytohaemagglutinin(PHA). Proliferation of lymphocytes was measured after 48 hours using³H-TdR incorporation as a marker for DNA synthesis.

Lymphocytes pretreated with the ethanol soluble fraction of emu oilshowed marked inhibition of PHA-induced lymphoproliferation (FIG. 16).This aspect has been repeated several times and similar results wereobtained reproducibly. Table 12 shows the results from a number ofexperiments which have examined the effect of ethanol extracts of Makinemu oil on lymphoproliferation. Using this assay system, the ethanolfractions from oils rendered at 40° C., 60° C. and 80° C. were tested.Interestingly, 60° C. and 80° C. oils were more active than 40° C. (FIG.17). TABLE 12 Summary of experiments examining the effects of variousethanol extractions of Makin emu oil on the lymphoproliferation responsein human T lymphocytes stimulated with PHA. A volume of 50 μl ofpurified T lymphocytes (4 × 10⁶/ml) was placed into a U-bottom well andan equal volume of ethanol or ethanol extract of Makin emu oil (final of1% whole emu oil equivalent) was added to the wells. The cells wereincubated at 37° C./5% CO₂/humid atmosphere for 30 min before 100 μl of5% AB serum or 2 μg/μl PHA (in 5% AB serum) was added to the wells. Thewells were then incubated at 37° C./5% CO₂/ humid atmosphere for 48hours. Six hours prior to harvesting, the cells were pulsed with 1 μCiof methyl-³H-thymidine. Incorporated radioactivity was measured using aβ counter. Experimental % Inhibition of Number LymphoproliferativeResponse 1. 84.3 ± 1.5 2. 84.2 ± 5.5 3. 85.0 ± 8.5 4.  99.9 ± 0.14 5. 99.75 ± 0.045 Mean ± sem 90.63 ± 3.76

Considering that Makin emu oil was found to be highly active ininhibiting DTH in comparison to G53 emu oil, the ethanol fractions fromthe two oil preparations were compared in their abilities to inhibit Tlymphocyte proliferation induced by PHA. The data presented in FIG. 18show that, while Makin emu oil caused >90% inhibition of the Tlymphocyte response, G53 emu oil produced only 50% inhibition of thisresponse.

12.6.2 Monocyte Function

Further experiments examined the effect of emu oil on cytokineproduction by T lymphocytes. As per lymphocyte proliferation assays, themononuclear leukocyte fraction was pretreated with the Makin emu oilethanol fraction and then stimulated with PHA. After 48 h incubation,the supernatants were assessed for levels of the cytokines, IFN-γ, TNF-βand IL-2 (FIG. 19).

The results showed that production of these cytokines, and in particularIFN-γ, was inhibited. Monocytes prepared as the adherent fraction ofmononuclear leukocytes were pretreated with Makin emu oil ethanolfraction and then stimulated with bacterial lipopolysaccharide (LPS).The effect on TNF-α production was assessed by measuring the cytokine inthe cultured treated or untreated monocytes. The results showed thatMakin ethanol fraction of emu oil was a poor inhibitor of LPS-inducedcytokine production (FIG. 20).

12.6.3 Neutrophil Adherence

Since neutrophils are the main proponents of acute inflammation,investigations were conducted as to whether the ethanol soluble emu oilfraction affected neutrophil functional responses essential forneutrophil tissue influx and whether accumulation of neutrophils atinflammatory sites requires the adhesion of neutrophils to theendothelium of blood vessels. This adhesion can be promoted byupregulating integrins on the neutrophil surface, as well as adhesionmolecules on the endothelial tissue.

In the first set of investigations, neutrophils were exposed to Makinemu oil ethanol fraction and then stimulated with phorbol myristateacetate (PMA). The results showed that the PMA-induced upregulation ofneutrophil adhesion to plastic surfaces was depressed by treatment withthis fraction of oil (FIG. 21).

In the second set of investigations, human umbilical vein endothelialcells were exposed to the Makin emu oil ethanol fraction. The cells werewashed and then stimulated with tumor necrosis factor (TNF) toupregulate the adhesion molecules. Fresh neutrophils were added to theendothelial cell monolayers and the degree of neutrophil adherence wasquantified. The (TNF) stimulated endothelial cells showed enhancedneutrophil adhesion and this was significantly reduced in endothelialcell cultures which had been pretreated with the emu oil (FIG. 22).

12.6.4 Neutrophil Chemotaxis

The ability of neutrophils to move into infection sites is dependent ontheir chemotactic response. In this investigation, the neutrophilchemotaxis response was quantified by measuring the degree of movementof neutrophils towards a chemotactic agent, the tripeptide FMLP. Thedata presented in FIG. 23 show that neutrophils, which had beenpretreated with Makin emu oil ethanol fraction, showed a poorchemotactic response.

12.7 Further Characterisation of the Anti-T Cell Activity of Emu Oil

Preliminary studies have also shown that some of the unsaturated fattyacids found in emu oil inhibit T lymphocyte and mononuclear cellresponses. Thus, our results show that 18:2ω6 is strongly inhibitorycompared with 18:1ω9, 18:0 and 18:2 (FIG. 24).

Since long chain fatty acids such as 18:2ω6 are suspected to beresponsible for the anti T cell effects, it was interesting to see ifthe fatty acid binding proteins in serum could prevent the activitypresent within the ethanol fraction. The lymphocytes were pretreatedwith the Makin emu oil ethanol fraction in the presence and absence of5% human blood group AB serum and then stimulated with PHA. The data inFIG. 25 show that serum could prevent the inhibitory effects of the emuoil ethanol fraction on T lymphocytes.

Chemical analysis of the ethanol fraction of Makin emu oil by GC showedthat the fatty acids were present in similar proportions to the wholeoil (Table 13). However, there was a small increase in 18:2ω6. TABLE 13GC fatty acid analyses of ethanol extract of Makin emu oil Fatty Acidwhole oil ethanol soluble extract  8:0  9:0 10:0 11:0 12:0 0.03 0.0813:0 14:0 0.42 0.59 15:0 0.07 0.08 dma 16:0 16:0 20.54 18.91 17:0 0.220.19 dma 18:0 18:0 11.14 7.95 20:0 0.21 0.18 22:0 0.03 24:0 Total Sets32.67 27.99 Trans 16:1 0.03 Trans 18:1w9 0.32 0.30 Trans 18:1w7 0.190.21 Trans 18:2 0.04 Totals Trans 0.58 0.51 11:1 12:1 13:1 14:1 0.090.16 15:1 16:1w9 0.15 0.18 16:1w7 2.95 4.06 17:1 18:1w9 47.88 48.5718:1w7 1.84 1.93 19:1 0.05 20:1w11 0.06 20:1w9 0.41 0.38 22:1w11 22:1w90.02 0.14 24:1w9 Total Monos 53.41 55.40 18:2w9 0.02 20:2w9 0.03 20:3w90.03 Total w9 48.53 49.26 Total w7 4.78 5.99 9,11 18:2 cLA 0.06 0.0510,12 18:2 cLA 18:2w6 11.95 14.69 18:3w6 0.04 0.04 20:2w6 0.10 0.1120:3w6 0.02 20:4w6 0.09 0.21 22:2w6 22:4w6 0.04 22:5w6 Total w6 12.2415.01 16:2w3 0.08 18:3w3 0.82 1.04 18:4w3 20:3w3 20:5w3 22:5w3 0.0422:6w3 Total w3 0.94 1.04

The ethanol soluble emu oil fraction was also subjected to TLC(analytical). This revealed seven bands (FIG. 26). Interestingly, band 3corresponded to 18:2ω6. A preparative run was also conducted and this isshown in FIG. 27, revealing 8 fractions. These fractions were thentested for the ability to inhibit lymphocyte proliferation. The resultsshowed that the major activity was associated with fractions 3,4 and 6,equalling fraction 3 (FIG. 28). The other fractions had much lessactivity. Interestingly, fraction 3 corresponds to 18:2ω6 mobility.

12.8 Anti-Inflammatory Properties of Emu Oil Triglyceride Fraction

The ethanol insoluble fraction contains primarily the triglyceridecomponent of the oil. This was tested for inhibiting activity on the DTHreaction. In these experiments, mice were treated with the triglyceridefraction of emu oil either 3 h prior to antigen challenge or 3 hpost-challenge. The DTH response was significantly reduced to a similarextent as the whole oil when the triglyceride fraction was appliedeither prior to or post antigen challenge (FIG. 29).

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1-31. (canceled)
 32. An assay system for grading a substance so as toassess, in a standardized manner, its anti-inflammatory activity, saidassay system comprising: (i) injection of a suitable antigen, into anappropriate body part of a mammal; (ii) either injection of apredetermined amount of said test substance into the same body part, ortopical application to said mammal of a predetermined amount of saidsubstance; (iii) measurement of the degree to which swelling which wouldotherwise result from injection of said antigen is reduced oralleviated; and (iv) comparing the activity of said test substance, asmeasured in step (iii), against the activity of a standard compoundhaving known anti-inflammatory characteristics, the activity of saidstandard compound having been measured by this same assay system ofsteps (i) to (iii), and having been used to generate a grading system tocompare the efficacy of various of the assessed substances.
 33. An assaysystem according to claim 32, wherein said substance is selected fromthe group consisting of oils, fats, organic solvent extracts of oils andfats, preparations comprising oils, preparations comprising fats,biologically active components of oils, and biologically activecomponents of fats.
 34. An assay system according to claim 33, whereinsaid substance is selected from the group consisting of animal oils andplant oils.
 35. An assay system according to claim 34 wherein the oil isselected from the group consisting of tea tree oil, flaxseed oil,linseed oil, borage oil and evening primrose oil; fish oils; and algal,microbial and fungi oils.
 36. An assay system according to claim 33,wherein said substance is emu oil or an ethanol extract of emu oil. 37.An assay system according to claim 32 wherein, in step (i), said antigenis injected intraperitoneally or into a footpad or ear of said mammal.38. An assay system according to claim 32, wherein said antigen isCarrageenan or sheep red blood cells.
 39. An assay system according toclaim 32 wherein, in step (ii), said substance is injectedintraperitoneally or applied topically.
 40. An assay system according toclaim 32 wherein steps (i) to (iv) are repeated, using serially reducingamounts of said substance.
 41. An assay system according to claim 40,wherein said substance is serially diluted in ethanol.
 42. An assaysystem for grading a substance so as to assess, in a standardizedmanner, its anti-inflammatory activity, said assay system comprising:(i) measurement of the activity of an in vitro preparation of T-cells,macrophages or neutrophils, or a cell line derived therefrom; (ii)addition of said substance to said preparation of T-cells, macrophagesor neutrophils, or said cell line derived therefrom; (iii) measurementof the change in activity of said preparation of T-cells, macrophages orneutrophils, or said cell line derived therefrom, following addition ofsaid substance in step (ii); and (iv) comparing the change in activity,as measured in step (iii), for said substance against the change inactivity for a standard compound having known anti-inflammatorycharacteristics, the change in activity for the standard compound havingbeen measured by this same assay system of steps (i) to (iii), andhaving been used to generate a grading system to compare the efficacy ofvarious of the assessed substances.
 43. An assay system according toclaim 42, wherein said substance is selected from the group consistingof oils, fats, organic solvent extracts of oils and fats, preparationscomprising oils, preparations comprising fats, biologically activecomponents of oils, and biologically active components of fats.
 44. Anassay system according to claim 43, wherein said substance is selectedfrom the group consisting of animal oils and plant oils.
 45. An assaysystem according to claim 44 wherein said oil is selected from the groupconsisting of tea tree oil, flaxseed oil, linseed oil, borage oil andevening primrose oil; fish oils; and algal, microbial and fungi oils.46. An assay system according to claim 43, wherein said substance is emuoil or an ethanol extract of emu oil.
 47. An assay system according toclaim 42, wherein said preparation is a preparation of T lymphocytes andsaid activity is lymphoproliferation.
 48. An assay system according toclaim 42, wherein said preparation is a preparation of T lymphocytes andsaid activity is production of cytokines.
 49. An assay system accordingto claim 48, wherein said cytokines are selected from the groupconsisting of interleukin-2, tumor necrosis factors and interferon-γ.50. An assay system according to claim 42, wherein said preparation is apreparation of neutrophils and said activity is chemotaxis.
 51. An assaysystem according to claim 42, wherein said preparation is a preparationof neutrophils and said activity is adherence to endothelial cells. 52.An assay system according to claim 42, wherein steps (i) to (iv) arerepeated, using serially reducing amounts of said substance.
 53. Anassay system according to claim 52, wherein said substance is seriallydiluted in ethanol.
 54. A method of preparing a therapeutically activeemu oil, comprising the heating of emu oil or tissue which contains emuoil to a temperature of at least 40° C. in order to obtain the activecomponents of the oil or tissue.
 55. The method of claim 54, wherein theheating temperature is in the range of about 40° to 100° C.
 56. Themethod of claim 54, wherein the heating temperature is in the range ofabout 60° to 80° C.
 57. The method of claim 54, wherein said temperatureis about 60° C.
 58. The method of claim 54, wherein said temperature isabout 80° C.
 59. The method of claim 54, wherein the heating temperatureis about 100° C.
 60. A method of preparing a therapeutically activefraction from emu oil or emu oil containing tissue, comprisingextracting said oil or tissue with an organic solvent to obtain theactive components of the oil or tissue.
 61. The method of claim 60,wherein the organic solvent is an alcohol.
 62. The method of claim 61,wherein the alcohol is ethanol.